Direct detection of genetic biomarkers in tissue and body fluids without complex target extraction and amplification processes can revolutionize nucleic acid-based diagnostics by enabling the use of this technology at the point-of-care. The development of point-of-care diagnostics is important to increase access to early treatment in underserved populations in low to middle income countries, which are disproportionally affected by infectious diseases and increasingly affected by certain types of cancer. The main obstacle to the development of such technologies is the low concentration of target sequences that makes this goal challenging. We report a method for direct detection of pathogen RNA in blood lysate using a bioassay using surface-enhanced Raman spectroscopy (SERS)-based detection assay that can be integrated in a “lab-in-a-stick” portable device. We could directly detect synthetic target with a limit of detection of 200 fM and, more importantly, we detected P. falciparum malaria parasite RNA directly in infected red blood cells lysate. Additionally, this paper will discuss the use of the developed assay for the identification of head and neck squamous cell carcinoma (HNSCC), which is an increasingly prevalent malignancy in low to middle income countries.
The development of rapid, easy-to-use and highly sensitive DNA detection methods has received increasing interest for medical diagnostics and research purposes. Our laboratory has developed several chip-based DNA biosensors including molecular sentinel-on-chip (MSC), multiplex MSC, and inverse molecular sentinel-on-chip (iMS-on-Chip). These sensors use surface-enhanced Raman scattering (SERS) plasmonic chips, functionalized with DNA probes for single-step DNA detection. The sensing mechanisms is based on the hybridization of target sequences and DNA probes, resulting in a displacement of a SERS reporter from the chip surface. This distance increase results in change in SERS signal intensity from the reporter, thus indicating the capture, and therefore the presence, of the target nucleic acid sequence. The nucleic acid probes and the SERS chip, which compose the sensing platform, were designed for single-step DNA detection. The target sequences are detected by delivery of a sample solutions on a functionalized chip and characterization of the SERS signals, after 1 - 2 hr incubation. These techniques avoid labeling of the target sequence or washing to remove unreacted components, making them easy-to-use and cost effective. The use of SERS chip for medical diagnostics was demonstrated by detecting genetic biomarkers for respiratory viral infection and the DNA of dengue virus 4.
The development of rapid, easy-to-use, cost-effective, high accuracy, and high sensitive DNA detection methods for molecular diagnostics has been receiving increasing interest. Over the last five years, our laboratory has developed several chip-based DNA detection techniques including the molecular sentinel-on-chip (MSC), the multiplex MSC, and the inverse molecular sentinel-on-chip (iMS-on-Chip). In these techniques, plasmonic surface-enhanced Raman scattering (SERS) Nanowave chips were functionalized with DNA probes for single-step DNA detection. Sensing mechanisms were based on hybridization of target sequences and DNA probes, resulting in a distance change between SERS reporters and the Nanowave chip’s gold surface. This distance change resulted in change in SERS intensity, thus indicating the presence and capture of the target sequences. Our techniques were single-step DNA detection techniques. Target sequences were detected by simple delivery of sample solutions onto DNA probe-functionalized Nanowave chips and SERS signals were measured after 1h - 2h incubation. Target sequence labeling or washing to remove unreacted components was not required, making the techniques simple, easy-to-use, and cost effective. The usefulness of the techniques for medical diagnostics was illustrated by the detection of genetic biomarkers for respiratory viral infection and of dengue virus 4 DNA.
Nucleic acid-based molecular diagnostics at the point-of-care (POC) and in resource-limited settings is still a challenge. We present a sensitive yet simple DNA detection method with single nucleotide polymorphism (SNP) identification capability. The detection scheme involves sandwich hybridization of magnetic beads conjugated with capture probes, target sequences, and ultrabright surface-enhanced Raman Scattering (SERS) nanorattles conjugated with reporter probes. Upon hybridization, the sandwich probes are concentrated at the detection focus controlled by a magnetic system for SERS measurements. The ultrabright SERS nanorattles, consisting of a core and a shell with resonance Raman reporters loaded in the gap space between the core and the shell, serve as SERS tags for ultrasensitive signal detection. Specific DNA sequences of the malaria parasite Plasmodium falciparum and dengue virus 1 (DENV1) were used as the model marker system. Detection limit of approximately 100 attomoles was achieved. Single nucleotide polymorphism (SNP) discrimination of wild type malaria DNA and mutant malaria DNA, which confers resistance to artemisinin drugs, was also demonstrated. The results demonstrate the molecular diagnostic potential of the nanorattle-based method to both detect and genotype infectious pathogens. The method's simplicity makes it a suitable candidate for molecular diagnosis at the POC and in resource-limited settings.
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